In fetal sheep, cardiomyocytes gradually cease dividing and become binucleated (terminal differentiation) at ~100 days of a 145 day gestational period. Once cardiac myocytes terminally differentiate they can no longer divide but they retain a remarkable capacity to enlarge. The hallmark of this maturation step is binucleation in sheep which occurs mostly before birth. We discovered that 3,3',5-tri-iodo-L-thyronine (T3), is a powerful inhibitor of proliferation in 135 day ovine cardiomyocytes in vitro. It appears that cortisol stimulates the conversion of the less potent thyroxine (T4) to the more potent T3 near term, causing T3 levels to rise-putting the brakes on cardiomyocyte proliferation. Because of its coincidental timing, T3 has become a primary candidate for being the most powerful regulator of the maturation of the myocardium. It may also terminate proliferation long before the heart has generated its optimal number of cardiomyocytes. Thus, T3 regulation has clinical relevance for the relatively common disease conditions when maternal thyroxine levels are outside the normal range. We request funds to study the role of thyroid hormone in regulating the maturation of the fetal myocardium in sheep. Aim 1: Determine the degree to which T3 suppresses proliferation and promotes binucleation/terminal differentiation of fetal cardiac myocytes in vivo. Hypothesis: T3 will depress the rates of proliferation of intact fetal ovine cardiomyocytes, increase the rate of terminal differentiation and stimulate cardiomyocyte maturation. Aim 2: Determine the developmental expression and temporal activation of key signaling proteins (MAPK & PI3K pathways) following exposure to T3 in fetal cardiac myocytes in vitro. Hypothesis: Both MAPK and PI3K are activated by T3, but proliferation is regulated by MAPK and its interaction with p21. The importance of the MAPK and PI3K signaling cascades in regulating proliferation under the influence of T3 will be evaluated by measuring the activation levels of ERK, AKT, mTOR and p70S6K, as well as key cell cycle proteins. Non-genomic pathways will also be evaluated. Aim 3: Determine the degree to which the fetal myocardium mal-adapts to right ventricular systolic load when T3 concentrations are elevated in vivo. Hypothesis: T3 treatment suppresses the normal proliferative response of cardiomyocytes to right ventricular (RV) systolic load and further stimulates the rate of binucleation and maturation of cardiomyocytes. Aim 4: Determine the degree to which cardiomyocyte growth and maturation are maintained during the early postnatal transition in fetuses that have been exposed to high T3 in utero. Hypothesis: The normal postnatal T3 surge will prevent suppressed cardiomyocyte numbers to regenerate during the immediate postnatal period, even in the presence of elevated levels of IGF-1. This study will determine the role of thyroid hormone in regulating the maturation of the myocardium before birth. Once completed the studies will indicate the degrees to which classical and non- classical signaling pathways regulate the T3 stimulated changes in cardiomyocyte behavior before birth. PUBLIC HEALTH RELEVANCE: This study will determine the relevance of fetal T3 levels in regulating the proliferation of working cardiomyocytes before birth. Because maternal thyroid hormones cross the placenta and influence fetal levels, maternal thyroid disease may seriously affect heart cardiomyocyte endowment. Low cardiomyocyte numbers could lead to a myocardium that is disadvantaged for the work it will perform in extrauterine life.